Antigen 43 associated with Escherichia coli membrane vesicles contributes to bacterial cell association and biofilm formation

Abstract

Bacterial membrane vesicles (MVs) are produced by all bacteria and contribute to numerous bacterial functions due to their ability to package and transfer bacterial cargo. In doing so, MVs have been shown to facilitate horizontal gene transfer, mediate antimicrobial activity, and promote biofilm formation. Uropathogenic Escherichia coli is a pathogenic Gram-negative organism that persists in the urinary tract of its host due to its ability to form persistent, antibiotic-resistant biofilms. The formation of these biofilms is dependent upon proteins such as Antigen 43 (Ag43), which belongs to the widespread Autotransporter group of bacterial surface proteins. In E. coli, the autotransporter Ag43 has been shown to contribute to bacterial cell aggregation and biofilm formation via self-association of Ag43 between neighboring Ag43-expressing bacteria. As MVs package bacterial proteins, we investigated whether MVs produced by E. coli contained Ag43, and the ability of Ag43-expressing MVs to facilitate cell aggregation and biofilm formation. We showed that Ag43 expressing E. coli produced MVs that contained Ag43 on their surface and had an enhanced ability to bind to E. coli bacteria. Furthermore, we demonstrated that the addition of Ag43-containing MVs to Ag43-expressing E. coli significantly enhanced biofilm formation. These findings reveal the contribution of MVs harboring autotransporters in promoting bacterial aggregation and enhancing biofilm formation, highlighting the impact of MVs and their specific composition to bacterial adaptation and pathogenesis.IMPORTANCEAutotransporter proteins are the largest family of outer membrane and secreted proteins in Gram-negative bacteria which contribute to pathogenesis by promoting aggregation, biofilm formation, persistence, and cytotoxicity. Although the roles of bacterial autotransporters are well known, the ability of bacterial membrane vesicles (MVs) naturally released from the surface of bacteria to contain autotransporters and their role in promoting virulence remains less investigated. Our findings reveal that MVs produced by E. coli contain the autotransporter protein Ag43. Furthermore, we show that Ag43-containing MVs function to enhance bacterial cell interactions and biofilm formation. By demonstrating the ability of MVs to carry functional autotransporter adhesins, this work highlights the importance of MVs in disseminating autotransporters beyond the bacterial cell membrane to ultimately promote cellular interactions and enhance biofilm development. Overall, these findings have significant implications in furthering our understanding of the numerous ways in which MVs can facilitate bacterial persistence and pathogenesis.

Keywords: Antigen 43; autotransporters; bacterial membrane vesicles; biofilm; outer membrane vesicles.

Fig 1

Characterization of E. coli Ag43^−ve and Ag43^+ve MVs. MVs isolated from (A) E. coli HEHA16 pBAD/MycHisA (Ag43^−ve) and (B) E. coli HEHA16 pBAD/MycHisA::agn43 (Ag43^+ve) were visualized by transmission electron microscopy. Scale bar = 100 nm. Images are representative of three biological replicates. (C) The number of Ag43^−ve and Ag43^+ve MVs produced per 10¹⁰ bacterial cells was determined by ZetaView Nanoparticle Tracking Analysis (NTA). Data are represented as the mean ± SEM of three biological replicates. *P < 0.05, unpaired t-test. (D) The number of viable Ag43^−ve and Ag43^+ve E. coli HEHA16 bacteria present in individual cultures from which MVs were isolated was determined by viable counts. Data show CFU/mL of individual cultures, and the mean ± SEM of three biological replicates. ns, not significant. (E) The size distribution of Ag43^−ve MVs and Ag43^+ve MVs was determined using ZetaView NTA. Data represent the mean of three biological replicates. (F) Quantification of the size distribution of Ag43^−ve MVs (squares; open bars) or Ag43^+ve MVs (circles; grey bars) by ZetaView NTA. Data are represented as the mean ± SEM of three biological replicates. *P < 0.05, unpaired t-test within each size range.

Fig 2

E. coli MVs contain Antigen 43. (A) MVs and MV-depleted supernatants (S/N) obtained from Ag43^+ve E. coli and Ag43^−ve E. coli cultures were separated using SDS-PAGE, and the presence of Ag43 was detected by Western blot using an anti-Ag43 antibody. Arrow indicates α43 (50 kDa) and purified Ag43 was used as a control (+ve). The image is representative of three biological replicates. (B) E. coli Ag43^−ve MVs and (C) Ag43^+ve MVs were labeled with rabbit anti-Ag43 antibodies, followed by an anti-rabbit antibody and protein-A-gold (PAG) and imaged by TEM. White arrows within the magnified image indicate the association of PAG with Ag43^+ve MVs. (D) Ag43^−ve MVs and (E) Ag43^+ve MVs were labeled with anti-rabbit antibodies and protein-A-gold (PAG) as a negative control. Images are representative of three biological replicates. Scale bar = 100 nm for all images and 50 nm in the magnified image.

Fig 3

Ag43 positive MVs interact with E. coli expressing Ag43. Interactions between E. coli HEHA16 bacteria and purified MVs were observed using confocal microscopy. Ag43^−ve or Ag43^+ve E. coli bacteria were labeled with DiI (shown as magenta) and Ag43^−ve or Ag43^+ve MVs were labeled with DiO (green) lipophilic stains and incubated together in various combinations as indicated for 3 h. (A) Ag43^−ve E. coli with PBS, (B) Ag43^−ve E. coli with Ag43^−ve MVs, (C) Ag43^−ve E. coli with Ag43^+ve MVs, (D) Ag43^+ve E. coli with PBS, (E) Ag43^+ve E. coli with Ag43^−ve MVs, and (F) Ag43^+ve E. coli with Ag43^+ve MVs. Colocalization is shown as white and indicated by the white arrows. Scale bar = 5 µm. Images are representative of three biological replicates. (G) Colocalization of E. coli and MVs as quantified by Manders coefficient between combinations of DiI labeled Ag43^+ve or Ag43^−ve E. coli bacteria and their DiO labeled MVs. Data are represented as the mean ± SEM of three biological replicates. ****P < 0.0001. One-way ANOVA with Tukey’s multiple comparisons test. (H) Cytofluorogram showing colocalization between DiI labeled Ag43^+veE. coli and DiO labeled Ag43^+ve MVs. Data are representative of three biological replicates with ≥3 fields of view per biological replicate.

Fig 4

Ag43^+ve MVs enhance the formation of E. coli bacterial biofilms. E. coli MS528 was grown for 44 h in the presence of either 10⁸ Ag43^−ve MVs (squares), 10⁸ Ag43^+ve MVs (circles), or PBS as a control (Control; triangles) to produce biofilms. The amount of biofilm formation was determined using a crystal violet biofilm assay. Biofilm formation was normalized to Ag43^+ve E. coli biofilm grown in the presence of PBS and represented as a percentage of biofilm growth. Data are represented as the mean ± SEM of four biological replicates. One-way ANOVA with Dunnett’s multiple comparison test, *P < 0.05. ns, not significant.

Fig 5

Model depicting interactions between E. coli bacteria and MVs facilitated by Ag43. Interactions between nearby E. coli bacteria are facilitated by the binding of Ag43 on the bacterial surface in an Ag43-Ag43 manner. In this study, we determined that the addition of Ag43^+ve MVs increased biofilm production by Ag43-expressing E. coli bacteria. Therefore, the presence of Ag43 on the surface of E. coli MVs may mediate greater number of interactions between Ag43 positive E. coli bacteria. Thus, Ag43-containing MVs may enhance biofilm formation by increasing the overall bacterial biofilm biomass.